Photocontrol of the GTPase activity of the small G protein K-Ras by using an azobenzene derivative

The small G protein Ras is a central regulator of cellular signal transduction processes, functioning as a molecular switch. Switch mechanisms utilizing conformational changes in nucleotide-binding motifs have been well studied at the molecular level. Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis and trans forms upon exposure to ultraviolet and visible light irradiation, respectively. Here, we introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the nucleotide-binding motif of Ras to regulate the GTPase activity by photoirradiation. We prepared four Ras mutants (G12C, Y32C, I36C, and Y64C) that have a single reactive cysteine residue in the nucleotide-binding motif. PAM was stoichiometrically incorporated into the cysteine residue of the mutants. The PAM-modified mutants exhibited reversible alterations in GTPase activity, nucleotide exchange rate, and interaction between guanine nucleotide exchange factor and Ras, accompanied by photoisomerization upon exposure to ultraviolet and visible light irradiation. The results suggest that incorporation of photochromic molecules into its nucleotide-binding motif enables photoreversible control of the function of the small G protein Ras.     

Regulation of Na(+)-K(+)-ATPase by cAMP-dependent protein kinase anchored on membrane via its anchoring protein

Na⁺-K⁺- ATPase -subunitsin basolateral membrane vesicles (BLMVs) purified from rat parotidglands were ³²P-labeled within 5 s by incubation with[-³²P]ATP at 37°C in the presence of cAMP, but nolabeling occurred without cAMP. Phosphorylation ofNa⁺-K⁺-ATPase was associated with a decrease inits activity. This -subunit phosphorylation disappeared when BLMVswere briefly incubated with cAMP and subsequent washing before theincubation with [-³²P]ATP, indicating that catalyticsubunit of protein kinase A (PKA) associated to BLMVs via binding withits RII regulatory subunit anchored on the membrane. In theabsence of cAMP, a PKA catalytic subunit readily reassociated with themembrane-bound RII subunit. HT-31 peptide inhibited theNa⁺-K⁺-ATPase phosphorylation by membrane-boundendogenous PKA, indicating an involvement of A-kinase anchoring protein(AKAP). AKAP-150 protein in BLMVs was shown by immunoblotting and anRII overlay assay and was coimmunoprecipitated by anti-RII antibody.These results show that Na⁺-K⁺-ATPase of ratparotid gland acinar cells is regulated in vivo by membrane-anchoredPKA via AKAP rather than by free cytosolic PKA.

     

S-adenosyl-L-homocysteine hydrolase is necessary for aldosterone-induced activity of epithelial Na(+) channels

The A6 cell line was used to study the role ofS-adenosyl-L-homocysteine hydrolase (SAHHase) inthe aldosterone-induced activation of the epithelial Na⁺channel (ENaC). Because aldosterone increases methylation of severaldifferent molecules, and because this methylation is associated withincreased Na⁺ reabsorption, we tested the hypothesis thataldosterone increases the expression and activity of SAHHase protein.The rationale for this work is that general methylation may be promotedby activation of SAHHase, the only enzyme known to metabolize SAH, apotent end-product inhibitor of methylation. Although aldosteroneincreased SAHHase activity, steroid did not affect SAHHase expression.Antisense SAHHase oligonucleotide decreased SAHHaseexpression and activity. Moreover, this oligonucleotide, as well as apharmacological inhibitor of SAHHase, decreased aldosterone-inducedactivity of ENaC via a decrease in ENaC open probability. The kineticsof ENaC in cells treated with antisense plus aldosterone were similarto those reported previously for the channel in the absence of steroid. This is the first report showing that active SAHHase, in part, increases ENaC open probability by reducing the transition rate fromopen states in response to aldosterone. Thus aldosterone-induced SAHHase activity plays a critical role in shifting ENaC from a gatingmode with short open and closed times to one with longer open andclosed times.

     

Regulation of cardiac Ca(2+) channel by extracellular Na(+)

Hyponatremia is a predictor of poor cardiovascular outcomes during acute myocardial infarction and in the setting of preexisting heart failure [1]. There are no definitive mechanisms as to how hyponatremia suppresses cardiac function. In this report we provide evidence for direct down-regulation of Ca(2+) channel current in response to low serum Na(+). In voltage-clamped rat ventricular myocytes or HEK 293 cells expressing the L-type Ca(2+) channel, a 15mM drop in extracellular Na(+) suppressed the Ca(2+) current by ～15%; with maximal suppression of ～30% when Na(+) levels were reduced to 100mM or less. The suppressive effects of low Na(+) on I(Ca), in part, depended on the substituting monovalent species (Li(+), Cs(+), TEA(+)), but were independent of phosphorylation state of the channel and possible influx of Ca(2+) on Na(+)/Ca(2+) exchanger. Acidification sensitized the Ca(2+) channel current to Na(+) withdrawal. Collectively our data suggest that Na(+) and H(+) may interact with regulatory site(s) at the outer recesses of the Ca(2+) channel pore thereby directly modulating the electro-diffusion of the permeating divalents (Ca(2+), Ba(2+)).     

Regulation of the epithelial Na(+) channel by extracellular acidification

The effect of extracellular acidification wastested on the native epithelial Na⁺ channel (ENaC) in A6epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-inducedfluctuation analysis in A6 epithelia and dual electrode voltage clampin oocytes. In A6 cells, a decrease of extracellular pH(pH_o) from 7.4 to 6.4 caused a slow stimulation of theamiloride-sensitive short-circuit current (I_Na)by 68.4 ± 11% (n = 9) at 60 min. This increaseof I_Na was attributed to an increase of openchannel and total channel (N_T) densities. Similar changes were observed with pH_o 5.4. The effects ofpH_o were blocked by buffering intracellularCa²⁺ with 5 µM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Inoocytes, pH_o 6.4 elicited a small transient increase of theslope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min)followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pH_ostimulate the native ENaC by increasing N_T butdo not appreciably affect ENaC expressed in Xenopus oocytes.These effects are distinct from those observed with decreasingintracellular pH with permeant buffers that are known to inhibit ENaC.

     

G protein modulates thyroid hormone-induced Na(+) channel activation in ventricular myocytes

To evaluate the effects of liothyronine (3,5,3'-triiodo-L-thyronine, T(3)) on Na(+) channel current (I(Na)) properties, I(Na) was recorded in adult guinea pig ventricular myocytes. T(3) (1 nM) acutely increased whole cell I(Na) and shifted the steady-state I(Na) inactivation curve dose dependently. When the pipette solution contained 100 microM GTP or GTPgammaS, the effect of T(3) on the whole cell I(Na) was increased two- to threefold. This effect was almost completely abolished by pertussis toxin preincubation. In the cell-attached patch, T(3) increased the open probability of single I(Na) by reducing the null probability. In the inside-out patch, T(3) effect was 10 times faster than that in whole cell and cell-attached patches while GTPgammaS was present and could be completely washed out. T(3) alone slightly increased the channel open probability by increasing the closed state to open state rate constant (k(CO)) and reducing the null probability. GTPgammaS exposure only increased the number of functional channels. T(3) and GTPgammaS synergistically enhanced the channel open probability 5.8 +/- 0.5-fold by increasing k(CO), decreasing the open state to absorbing inactivated state rate constant, and greatly reducing the null probability. These results demonstrate that T(3) acts on the cytosolic side of the membrane and acutely activates I(Na). Pertussis toxin-sensitive G protein modulation greatly magnifies the T(3) effects on the channel kinetics and null probability, thereby increasing the channel open probability.     

Regulation of Na(+)-coupled glucose carrier SGLT1 by human papillomavirus 18 E6 protein

Tumor cells utilize preferably glucose for energy production. They accomplish cellular glucose uptake in part through Na⁺-coupled glucose transport mediated by SGLT1 (SLC5A1). This study explored the possibility that the human papillomavirus 18 E6 protein HPV18 E6 (E6) participates in the stimulation of SGLT1 activity. E6 is one of the two major oncoproteins of high-risk human papillomaviruses, which are the causative agent for cervical carcinoma. According to Western blotting, SGLT1 is expressed in the HPV18-positive cervical carcinoma cell line HeLa. To explore whether E6 affects SGLT1 activity, SGLT1 was expressed in Xenopus oocytes with and without E6 and electrogenic glucose transport determined by dual electrode voltage clamp. In SGLT1-expressing oocytes, but not in oocytes injected with water or expressing E6 alone, glucose triggered a current (I_g). I_g was significantly increased by coexpression of E6 but not by coexpression of E2. According to chemiluminescence and confocal microscopy, coexpression of E6 significantly increased the SGLT1 protein abundance in the cell membrane. The decay of I_g following inhibition of carrier insertion by Brefeldine A (5 μM) was not significantly affected E6 coexpression. Accrodingly, E6 was not effective by increasing carrier protein stability in the membrane. In conclusion, HPV18 E6 oncoprotein participates in the upregulation of SGLT1.     

Regulation of the epithelial Na+ channel by the protein kinase CK2

CK2 is a ubiquitous, pleiotropic, and constitutively active Ser/Thr protein kinase that controls protein expression, cell signaling, and ion channel activity. Phosphorylation sites for CK2 are located in the C terminus of both beta- and gamma-subunits of the epithelial Na(+) channel (ENaC). We examined the role of CK2 on the regulation of both endogenous ENaC in native murine epithelia and in Xenopus oocytes expressing rENaC. In Ussing chamber experiments with mouse airways, colon, and cultured M1-collecting duct cells, amiloride-sensitive Na(+) transport was inhibited dose-dependently by the selective CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). In oocytes, ENaC currents were also inhibited by TBB and by the structurally unrelated inhibitors heparin and poly(E:Y). Expression of a trimeric channel lacking both CK2 sites (alphabeta(S631A)gamma(T599A)) produced a largely attenuated amiloride-sensitive whole cell conductance and rendered the mutant channel insensitive to CK2. In Xenopus oocytes, CK2 was translocated to the cell membrane upon expression of wt-ENaC but not of alphabeta(S631A)gamma(T599A)-ENaC. Phosphorylation by CK2 is essential for ENaC activation, and to a lesser degree, it also controls membrane expression of alphabetagamma-ENaC. Channels lacking the Nedd4-2 binding motif in beta-ENaC (R561X, Y618A) no longer required the CK2 site for channel activity and siRNA-knockdown of Nedd4-2 eliminated the effects of TBB. This implies a role for CK2 in inhibiting the Nedd4-2 pathway. We propose that the C terminus of beta-ENaC is targeted by this essential, conserved pleiotropic kinase that directs its constitutive activity toward many cellular protein complexes.     

Regulation of Na(+) pump expression by vascular smooth muscle cells

The Na(+) pump and its regulation is important for maintaining membrane potential and transmembrane Na(+) gradient in all mammalian cells and thus is essential for cell survival and function. Vascular smooth muscle cells (VSMC) have a relatively low number of pump sites on their membrane compared with other cells. We wished to determine the mechanisms for regulating the number of pump sites in these cells. We used canine saphenous vein VSMC cultured in 10% serum and passaged one time. These cells were subcultured in 5% serum media with low K(+) (1 mM vs. control of 5 mM), and their pump expression was assessed. These VSMC upregulated their pump sites as early as 4 h after treatment (measured by [(3)H]ouabain binding). At this early time point, there was no detectable increase in protein expression of either alpha(1)- or beta(1)-subunits of the pump shown by Western blots. When the cells were treated with the phosphoinositide 3-kinase (PI-3-K) inhibitor LY-294002 (which is known to inhibit cytoplasmic transport processes) in low-K(+) media, the pump site upregulation was inhibited. These data suggest that the low-K(+)-induced upregulation of Na(+) pump number can occur by translocation of preformed pumps from intracellular stores.     

Regulation of ENA1 Na(+)-ATPase gene expression by the Ppz1 protein phosphatase is mediated by the calcineurin pathway

Saccharomyces cerevisiae strains lacking the Ppz1 protein phosphatase are salt tolerant and display increased expression of the ENA1 Na⁺-ATPase gene, a major determinant for sodium extrusion, while cells devoid of the similar Ppz2 protein do not show these phenotypes. However, a ppz1 ppz2 mutant displays higher levels of ENA1 expression than the ppz1 strain. We show here that the increased activity of the ENA1 promoter in a ppz1 ppz2 mutant maps to two regions: one region located at −751 to −667, containing a calcineurin-dependent response element (CDRE), and one downstream region (−573 to −490) whose activity responds to intracellular alkalinization. In contrast, the increased ENA1 expression in a ppz1 mutant is mediated solely by an intact calcineurin/Crz1 signaling pathway, on the basis that (i) this effect maps to a single region that contains the CDRE and (ii) it is blocked by the calcineurin inhibitor FK506, as well as by deletion of the CNB1 or CRZ1 gene. The calcineurin dependence of the increased ENA1 expression of a ppz1 mutant would suggest that Ppz1 could negatively regulate calcineurin activity. In agreement with this notion, a ppz1 strain is calcium sensitive, and this mutation does not result in a decrease in the calcium hypertolerance of a cnb1 mutant. It has been shown that ENA1 can be induced by alkalinization of the medium and that a ppz1 ppz2 strain has a higher intracellular pH. However, we present several lines of evidence that show that the gene expression profile of a ppz1 mutant does not involve an alkalinization effect. In conclusion, we have identified a novel role for calcineurin, but not alkalinization, in the control of ENA1 expression in ppz1 mutants.     

Rho small GTPases activate the epithelial Na(+) channel

Small G proteins in the Rho family are known to regulate diverse cellular processes, including cytoskeletal organization and cell cycling, and more recently, ion channel activity and activity of phosphatidylinositol 4-phosphate 5-kinase (PI(4)P 5-K). The present study investigates regulation of the epithelial Na(+) channel (ENaC) by Rho GTPases. We demonstrate here that RhoA and Rac1 markedly increase ENaC activity. Activation by RhoA was suppressed by the C3 exoenzyme. Inhibition of the downstream RhoA effector Rho kinase, which is necessary for RhoA activation of PI(4)P 5-K, abolished ENaC activation. Similar to RhoA, overexpression of PI(4)P 5-K increased ENaC activity suggesting that production of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in response to RhoA-Rho kinase signaling stimulates ENaC. Supporting this idea, inhibition of phosphatidylinositol 4-kinase, but not the RhoA effector phosphatidylinositol 3-kinase and MAPK cascades, markedly attenuated RhoA-dependent activation of ENaC. RhoA increased ENaC activity by increasing the plasma membrane levels of this channel. We conclude that RhoA activates ENaC via Rho kinase and subsequently activates PI(4)P 5-K with concomitant increases in PI(4,5)P(2) levels promoting channel insertion into the plasma membrane.     

Regulation of mitogen-activated protein kinases in cardiac myocytes through the small G protein Rac1

Small guanine nucleotide-binding proteins of the Ras and Rho (Rac, Cdc42, and Rho) families have been implicated in cardiac myocyte hypertrophy, and this may involve the extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and/or p38 mitogen-activated protein kinase (MAPK) cascades. In other systems, Rac and Cdc42 have been particularly implicated in the activation of JNKs and p38-MAPKs. We examined the activation of Rho family small G proteins and the regulation of MAPKs through Rac1 in cardiac myocytes. Endothelin 1 and phenylephrine (both hypertrophic agonists) induced rapid activation of endogenous Rac1, and endothelin 1 also promoted significant activation of RhoA. Toxin B (which inactivates Rho family proteins) attenuated the activation of JNKs by hyperosmotic shock or endothelin 1 but had no effect on p38-MAPK activation. Toxin B also inhibited the activation of the ERK cascade by these stimuli. In transfection experiments, dominant-negative N17Rac1 inhibited activation of ERK by endothelin 1, whereas activated V12Rac1 cooperated with c-Raf to activate ERK. Rac1 may stimulate the ERK cascade either by promoting the phosphorylation of c-Raf or by increasing MEK1 and/or -2 association with c-Raf to facilitate MEK1 and/or -2 activation. In cardiac myocytes, toxin B attenuated c-Raf(Ser-338) phosphorylation (50 to 70% inhibition), but this had no effect on c-Raf activity. However, toxin B decreased both the association of MEK1 and/or -2 with c-Raf and c-Raf-associated ERK-activating activity. V12Rac1 cooperated with c-Raf to increase expression of atrial natriuretic factor (ANF), whereas N17Rac1 inhibited endothelin 1-stimulated ANF expression, indicating that the synergy between Rac1 and c-Raf is potentially physiologically important. We conclude that activation of Rac1 by hypertrophic stimuli contributes to the hypertrophic response by modulating the ERK and/or possibly the JNK (but not the p38-MAPK) cascades.     

Regulation of fish gill Na(+)-K(+)-ATPase by selective sulfatide-enriched raft partitioning during seawater adaptation

Na(+)-K(+)-ATPase is arguably the most important enzyme in the animal cell plasma membrane, but the role of the membrane in its regulation is poorly understood. We investigated the relationship between Na(+)-K(+)-ATPase and membrane microdomains or "lipid rafts" enriched in sulfatide (sulfogalactosylceramide/SGC), a glycosphingolipid implicated as a cofactor for this enzyme, in the basolateral membrane of rainbow trout gill epithelium. Our studies demonstrated that when trout adapt to seawater (33 ppt), Na(+)-K(+)-ATPase relocates to these structures. Arylsulfatase-induced desulfation of basolateral membrane SGC prevented this relocation and significantly reduced Na(+)-K(+)-ATPase activity in seawater but not freshwater trout. We contend that Na(+)-K(+)-ATPase partitions into SGC-enriched rafts to help facilitate the up-regulation of its activity during seawater adaptation. We also suggest that differential partitioning of Na(+)-K(+)-ATPase between these novel SGC-enriched regulatory platforms results in two distinct, physiological Na(+) transport modes. In addition, we extend the working definition of cholesterol-dependent raft integrity to structural dependence on the sulfate moiety of SGC in this membrane.     

Inhibition of Na(+)-independent H+ pump by Na(+)-induced changes in cell Ca2+

Apical membrane H+ extrusion in the renal outer medullary collecting duct, inner stripe, is mediated by a Na(+)-independent H+ pump. To examine the regulation of this transporter, cell pH and cell Ca2+ were measured microfluorometrically in in vitro perfused tubules using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2, respectively. Apical membrane H+ pump activity, assayed as cell pH recovery from a series of acid loads (NH3/NH+4 prepulse) in the total absence of ambient Na+, initially occurred at a slow rate (0.06 +/- 0.02 pH units/min), which was not sufficient to account for physiologic rates of H+ extrusion. Over 15-20 min after the initial acid load, the rate of Na(+)-independent cell pH recovery increased to 0.63 +/- 0.09 pH units/min, associated with a steady-state cell pH greater than the initial pre-acid load cell pH. This pattern suggested an initial suppression followed by a delayed activation of the apical membrane H+ pump. Replacement of peritubular Na+ with choline or N-methyl-D-glucosamine resulted in an initial spike increase in cell Ca2+ followed by a sustained increase in cell Ca2+. The initial rate of Na(+)-independent cell pH recovery could be increased by elimination of the Na+ removal-induced sustained cell Ca2+ elevation by: (a) performing studies in the presence of 135 mM peritubular Na+ (1 mM peritubular amiloride used to inhibit basolateral membrane Na+/H+ antiport); (b) clamping cell Ca2+ low with dimethyl-BAPTA, an intracellular Ca2+ chelating agent; or (c) removal of extracellular Ca2+. Cell acidification induced a spike increase in cell Ca2+. The late acceleration of Na(+)-independent cell pH recovery was independent of Na+ removal and of the method used to acidify the cell, but was eliminated by prevention of the cell Ca2+ spike and markedly delayed by the microfilament-disrupting agent, cytochalasin B. This study demonstrates that peritubular Na+ removal results in a sustained elevation in cell Ca2+, which inhibits the apical membrane H+ pump. In addition, rapid cell acidification associated with a spike increase in cell Ca2+ leads to a delayed activation of the H+ pump. Thus, cell Ca2+ per se, or a Ca(2+)-activated pathway, can modulate H+ pump activity.     

Differences in the Regulation of K-Ras and H-Ras Isoforms by Monoubiquitination

Ras GTPases are signaling switches that control critical cellular processes including gene expression, differentiation, and apoptosis. The major Ras isoforms (K, H, and N) contain a conserved core GTPase domain, but have distinct biological functions. Among the three Ras isoforms there are clear differences in post-translational regulation, which contribute to differences in localization and signaling output. Modification by ubiquitination was recently reported to activate Ras signaling in cells, but the mechanisms of activation are not well understood. Here, we show that H-Ras is activated by monoubiquitination and that ubiquitination at Lys-117 accelerates intrinsic nucleotide exchange, thereby promoting GTP loading. This mechanism of Ras activation is distinct from K-Ras monoubiquitination at Lys-147, which leads to impaired regulator-mediated GTP hydrolysis. These findings reveal that different Ras isoforms are monoubiquitinated at distinct sites, with distinct mechanisms of action, but with a common ability to chronically activate the protein in the absence of a receptor signal or oncogenic mutation.     

Regulation of the Na(+)-dependent and the Na(+)-independent polyamine transporters in renal epithelial cells (LLC-PK1)

We have studied the regulation of the Na(+)-dependent and Na(+)-independent polyamine transport pathways in the renal LLC-PK1 cell line. Most of the experiments were performed in the presence of 5 mM DL-2-difluoromethylornithine (DFMO) in order to inhibit the cellular synthesis of polyamines. The activity of both transporters as measured by putrescine uptake was increased by growth-promoting stimuli and decreased by exogenous polyamines. The time course of the increase in uptake activity induced by fetal calf serum could be fitted by a single exponential, and the process was three times faster for the Na(+)-dependent than for the Na(+)-independent transporter. Maximum activity was reached after more than 24 h. This increase could be inhibited by actinomycin D and by cycloheximide. Other growth-promoting stimuli, such as subconfluent cell density, as well as growth factors also induced an increase in the transport activity. Particularly, there was a marked stimulation of the Na(+)-dependent pathway by epidermal growth factor in combination with insulin. On the other hand, the transport activity decayed very rapidly upon addition of exogenous polyamines (t1/2 less than 60 min). The diamine putrescine was much less effective in this respect than the polyamines spermidine and spermine. The non-metabolizable substrate methylglyoxal bis(guanylhydrazone) did not induce a decay of the transport activity, but it protected the Na(+)-dependent pathway against the polyamine-induced decay. Inhibition of the protein synthesis by cycloheximide did not induce a rapid decrease of the transport activity; neither did it affect the polyamine-induced decay. These observations suggest that this polyamine-induced decay is not owing to an inhibitory effect on the rate of synthesis of the transporters, but rather to a degradation or an inactivation of the transporters. The polyamine-induced decay slowed down at lower cell density. This effect was particularly pronounced for the Na(+)-dependent transporter. Since the uptake of polyamines was increased at low cell density, the decreased rate of decay in this condition pleads against a simple mechanism of transinhibition by the substrate. In conclusion, both transport pathways were similarly affected by the regulatory parameters, but the Na(+)-dependent transporter was more rapidly and more effectively regulated. The numerous interacting regulatory steps furthermore suggest a physiological role for these transporters, such as an involvement in urinary polyamine disposal.     

Regulation of the cardiac Na(+)-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca(2+)-binding domain

The sarcolemmal Na(+)-Ca2+ exchanger is regulated by intracellular Ca2+ at a high affinity Ca2+ binding site separate from the Ca2+ transport site. Previous data have suggested that the Ca2+ regulatory site is located on the large intracellular loop of the Na(+)-Ca2+ exchange protein, and we have identified a high-affinity 45Ca2+ binding domain on this loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Journal of Biological Chemistry. 269:22847-22852). We now use electrophysiological and mutational analyses to further define the Ca2+ regulatory site. Wild-type and mutant exchangers were expressed in Xenopus oocytes, and the exchange current was measured using the inside- out giant membrane patch technique. Ca2+ regulation was measured as the stimulation of reverse Na(+)-Ca2+ exchange (intracellular Na+ exchanging for extracellular Ca2+) by intracellular Ca2+. Single-site mutations within two acidic clusters of the Ca2+ binding domain lowered the apparent Ca2+ affinity at the regulatory site from 0.4 to 1.1-1.8 microM. Mutations had parallel effects on the affinity of the exchanger loop for 45Ca2+ binding (Levitsky et al., 1994) and for functional Ca2+ regulation. We conclude that we have identified the functionally important Ca2+ binding domain. All mutant exchangers with decreased apparent affinities at the regulatory Ca2+ binding site also have a complex pattern of altered kinetic properties. The outward current of the wild-type Na(+)-Ca2+ exchanger declines with a half time (th) of 10.8 +/- 3.2 s upon Ca2+ removal, whereas the exchange currents of several mutants decline with th values of 0.7-4.3 s. Likewise, Ca2+ regulation mutants respond more rapidly to Ca2+ application. Study of Ca2+ regulation has previously been possible only with the exchanger operating in the reverse mode as the regulatory Ca2+ and the transported Ca2+ are then on opposite sides of the membrane. The use of exchange mutants with low affinity for Ca2+ at regulatory sites also allows demonstration of secondary Ca2+ regulation with the exchanger in the forward or Ca2+ efflux mode. In addition, we find that the affinity of wild-type and mutant Na(+)-Ca2+ exchangers for intracellular Na+ decreases at low regulatory Ca2+. This suggests that Ca2+ regulation modifies transport properties and does not only control the fraction of exchangers in an active state.